Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in viv...

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Veröffentlicht in:	PloS one 2015-10, Vol.10 (10), p.e0139695-e0139695
Hauptverfasser:	Zhong, Nan, Loppnau, Peter, Seitova, Alma, Ravichandran, Mani, Fenner, Maria, Jain, Harshika, Bhattacharya, Anandi, Hutchinson, Ashley, Paduch, Marcin, Lu, Vincent, Olszewski, Michal, Kossiakoff, Anthony A, Dowdell, Evan, Koide, Akiko, Koide, Shohei, Huang, Haiming, Nadeem, Vincent, Sidhu, Sachdev S, Greenblatt, Jack F, Marcon, Edyta, Arrowsmith, Cheryl H, Edwards, Aled M, Gräslund, Susanne
Format:	Artikel
Sprache:	eng
Schlagworte:	Antibodies Antibody Formation - physiology Antigens Antigens - immunology Biochemistry Biomedical research Biotinylation Chromatin Cloning, Molecular Comparative analysis Consortia Cytochrome E coli Enzymes Escherichia coli Genetic aspects Genomics Humans Immunoglobulin Fab Fragments - immunology Immunoglobulin G Immunoglobulins Immunoprecipitation In vivo methods and tests Lysates Mammalian cells Mammals Molecular biology Peptide Library Peptides Phage display Phages Physiological aspects Proteins Proteomics Recombinant Proteins - immunology RNA polymerase Stem cells Workflow Yield
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Beschreibung
Zusammenfassung:	We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0139695